Nucleotide binding to IAF-labelled Na+/K(+)-ATPase measured by steady state fluorescence quenching by TNP-ADP.
Nucleotide binding to 5-iodoacetamidofluorescein (IAF) labelled Na+/K(+)-ATPase was measured by steady state fluorescence quenching of the fluorescein label via energy transfer to trinitrophenyl (TNP) labelled nucleotide. TNP-nucleotides are valuable probes of nucleotide binding to ATPases. Interpretation of these and other experiments in our laboratory using TNP-nucleotides with the Na+/K(+)-ATPase rely on having a good model for the interaction of TNP-nucleotide with the enzyme. Sets of fluorescence quenching curves obtained by titrating the enzyme with TNP-ADP in the presence of various concentrations of ADP could not be adequately modelled using a simple model with a single nucleotide binding site. Therefore, we compare various models which allow for additional TNP-nucleotide binding to the enzyme. In the two-site model, the additional binding is to a second specific site for which TNP-nucleotide and unlabelled nucleotide compete. In two other models, the additional binding (in one case saturable, and in the other case non-saturable) of TNP-nucleotide is not blocked by or affected by unlabelled nucleotide, and is, therefore, referred to as non-specific binding of the TNP-nucleotide. The goal of this work is to determine which of the distinctly different physical pictures associated with these models most accurately describes the interaction of TNP-nucleotide with the enzyme. We find that the interaction of TNP-ADP with IAF-labelled Na+/K(+)-ATPase is best described by a model in which there are two classes of binding: TNP-ADP and ADP compete for a specific binding site with dissociation binding constants of 0.13 microM for TNP-ADP and 2.0 microM for ADP; and non-saturable non-specific binding of TNP-ADP.
Hellen
E H
EH
Department of Physics and Astronomy, University of North Carolina at Greensboro 27412, USA.
Pratap
P R
PR
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004285:Dogs; D005452:Fluoresceins / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D009711:Nucleotides; D000244:Adenosine Diphosphate; C015296:5-iodoacetamidofluorescein; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000254:Sodium-Potassium-Exchanging ATPase